CNPS isolation and cultivation In the present study, we successfully isolated CNPs-like entities from both supragingival and subgingival dental plaque samples originating from patients afflicted with periodontal disease. Notably, a greater abundance of CNPs was observed within the subgingival plaque, thereby prompting an exploration into the potential association between CNPs and periodontitis. Plausible explanations for this phenomenon may exist in the content of inorganic components in plaque[30], notably encompassing calcium and phosphate ions, which could provide the growth impetus for CNPs formation and advantage to initiate self-replication and form an HA shell process. Besides, the alkaline products metabolized by nitrogen compounds are important factors to regulate the pH of dental plaque[31]. Processes such as the urea metabolic pathway and arginine decomposition pathway contribute to pH modulation within the plaque, thereby engendering elevated concentrations of inorganic ions such as calcium and phosphorus, consequently fostering a favourable environment for CNP proliferation. [32, 33]. Moreover, it is crucial to consider the intricacies of the microecological habitat within the subgingival plaque, characterized by a predominantly anaerobic environment often localized within the gingival pocket [34]. The oxidation-reduction potential was low, which made cleaning difficult, and such an environment may be more conducive to CNP colonization[35].
In terms of precipitation morphology, four aggregation forms of CNPs in dental plaque were observed from week 4: clinging to the bottom of the test tube, partially clinging and partial suspension, complete suspension upon light oscillation, and multi-site aggregation and clinging. Such an interesting phenomenon leads to speculation of the appearance of suspended matter with unstable morphologies is likely to be an intermediate CNPs form that appears without fully reaching the solid-phase transformation conditions.
By observing the growth curves, it can be seen that the lag and logarithmic growth phases of CNPs, but there was no significant trend of entering the stationary and decline phases, and growth increased continuously over the culture time. This is consistent with the growth rates mentioned in the literature[7, 36]. The OD values were higher in the SUBDP group compared to the SUPDP group from week 4, suggesting that the SUBDP group may have a higher number of isolated CNPs with greater agglomeration ability.
ALP expression level in CNPs ALPs is a homodimeric enzyme that was discovered to be one of the first key actors in the osteogenesis process. [37]. It is thought to promote the mineralization process by hydrolysing inorganic pyrophosphate (PPi) and raising the concentration of inorganic phosphates[38]. To acknowledge, although pathological calcification was thought to be a completely degenerative, uncontrolled process, the mechanism should be similar to physiological calcification[39]. Some studies have concluded that overexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelium leads to arterial calcification, while TNAP inhibition protects against it in mouse models[40, 41]. Accordingly, ALPs are recognized as involved in the pathogenesis of ectopic calcification[42].
Based on these insights and considering ALP's roles in biomineralization and pathological calcification, we hypothesize ALP's participation in CNP-induced mineralization as nucleation cores using environmental components. Consequently, we studied ALP expression in the supernatant during isolated sample culture.
ALP levels showed a decreasing trend over the culture time. The ALP activity level in the CNPs-positive group existed varying degrees of differences compared with the culture-negative group and control group in both supragingival and subgingival dental plaque samples. These findings lead us to tentatively propose that ALP's involvement in CNP formation might occur once a certain threshold is reached. Several considerations regarding the source of ALP come to:
a). CNPs might inherently secrete ALP during growth, thereby contributing to mineralization. However, the challenge arises from the inability to extract CNPs' nucleic acids, casting uncertainty on their classification as living organisms.
b). ALP derived from FBS. The presence of ALP in the control group suggests its potential occurrence in the FBS in the culture medium. Additionally, the ALP level was the same as before or even slightly increased after fluid replacement (e.g., week 3), in both SUPDP and SUBDP groups. FBS is a main supplement used in cell culture. It encompasses diverse components such as extracellular vesicles, lipoproteins, protein aggregates, and RNA. However, it's crucial to acknowledge that FBS composition can vary among different batches, potentially introducing contaminants like endotoxins, mycoplasma, viruses, and prion proteins, which might influence experimental outcomes[43]. Whether the inclusion of CNPs isolates triggers alterations in FBS-based ALP levels remains unknown.
c). Effects from the microenvironment around the sampling site. Salivary/gingival crevicular fluid (GCF)-ALP emerges as a biomarker for periodontitis, displaying elevated expression in this pathological context. Fibroblasts, polymorphonuclear leukocytes, and various oral bacteria, particularly Gram-negative microorganisms in subgingival plaque, contribute to increased ALP levels in response to periodontal tissue damage [44, 45]. Anatomical regions exposed to elevated ALP concentrations could potentially foster CNP formation. Furthermore, this might elucidate the higher isolation rate of CNPs from subgingival plaque compared to supragingival plaque.
Morphological Observation of CNPs Regarding the morphology, the CNPs isolated from plaque were similar to those in previous literature[7, 19, 46], which were round and small granular under light microscopy. HE and ARS staining suggested the presence of Ca salt components. Under TEM, it appeared as round, oval, and ellipsoid particles. The surrounding high-density linear shadow may indicate encasement of the calcified shell, and finer scattered particles were observed between the particles. A potential explanation for this phenomenon is that CNPs were using inorganic ions in the surrounding environment to form a mineralized shell.
CNP Chemical Composition Analysis The chemical composition of CNPs was investigated. The measured Ca/P ratio of CNPs, 0.6753, differed from that of both HA and dental calculus, which stand at 1.67[47]. This divergence could stem from a relatively low early-stage Ca concentration during CNP formation, possibly indicating an incomplete maturation process. Stability was gradually achieved with increased Ca uptake, which was consistent with our macroscopic observation that some samples became semi-suspended or suspended upon shaking.
FTIR analysis showed significant functional groups common to HA and dental calculus, except for the methyl group. According to the protein-mineral theory, Peng [19] identified fetuin-A, albumin, and apolipoprotein A1 as primary protein components within CNPs. The presence of methyl group indicates the presence of organic matter, after ruling out the influence of compounds such as alcohol on the experimental procedure. It is therefore likely that this methyl group is derived from some protein component of the culture process, and its specific contributors need to be further studied.
The crystal morphology of CNPs examined by XRD. Consistency in peak patterns across six samples indicates the stability of CNPs' primary chemical components. Database comparison revealed the predominant presence of HA and tricalcium phosphate, mirroring dental calculus's chemical makeup. Consistency in peak patterns across six samples indicates the stability of CNPs' primary chemical components. Database comparison revealed the predominant presence of HA and tricalcium phosphate, mirroring dental calculus's chemical makeup. This demonstrates that CNPs produce compounds similar to dental calculus during the culture phase.
Limitations and Prospects In our preliminary study, we've successfully established an in vitro isolation and culture method for CNPs derived from dental plaque. Simultaneously, we also analyzed and made inferences in response to the experimental results. Nevertheless, certain limitations persist. CNPs have been characterized only in terms of morphology and chemical composition, without sufficient evidence of the origin of their core components. At present, mineral-organic nanoparticles are widely accepted in academic circles, with fetuin-A thought to function as nucleation seeds that expand through ion sedimentation to produce bigger amorphous nanoparticles[20, 48]. Consequently, exploring the levels and types of proteins in their components should be an urgent issue to be addressed due to the failure to validate the nucleic acid sequences.
On the other hand, in addition to inducing pathological calcification formation, the cytotoxic effect of CNPs on periodontal tissues and the symbiotic state with periodontal pathogenic bacteria in the periodontal microbial ecosystems represent directions worth exploring.